Transglutaminase 2 (TG2) is a member of the human transglutaminase family of enzymes, which is abundantly expressed in various tissues and is found in both intra- and extracellular locations (Lorand and Graham, 2003 Nat. Rev. Mol. Biol. 4, 140-156). It possesses the catalytic activity of crosslinking of glutamine sidechains on substrate peptides or proteins with biogenic small molecule or protein-bound amines, which is subject to elaborate posttranslational regulation. TG2 has been attributed with various biological functions (for reviews, see e.g. (Iismaa et al., 2009 Physiol. Rev. 89, 991-1023; Mehta et al., 2005; Nurminskaya and Belkin, 2012 Int. Rev. Cell Mol. Biol. 294, 1-97) and it has been implicated in the pathogenesis of a broad range of human diseases, particularly inflammatory disorders.
Examples of such disorders are sepsis, where studies on TG2−/− mice suggest a pathogenic role for TG2 in the development of endotoxic shock (Falasca et al., 2008 J. Immunol. 180, 2616-2624). Similarly, the crosslinking activity of TG2 has been implicated in the development of vascular calcification and renal fibrosis (Chen et al., 2013 Am. J. Nephrol. 37, 191-198). Additionally, it might play a pathogenic role in ischemic reperfusion injury, as suggested by studies involving genetic ablation or pharmacologic inhibition of TG2 (Kim et al., 2010 Biochem. Biophys. Res. Commun. 403, 479-484; Shin et al., 2008 Biochem. Biophys. Res. Commun. 365, 509-514)
Such reports have motivated the development of TG2 inhibitors as tools for TG2 research. One such class of compounds is based on the mildly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) moiety. Early studies in the literature (Castelhano et al., 1988 Biochem. Biophys. Res. Commun. 365, 509-514; Killackey et al., 1989 Mol Pharmacol. 35, 701-706) and previous studies from our lab (Choi et al., 2005 Chem. Biol. 12, 469-475; Watts et al., 2006 J. Med. Chem. 49, 7493-7501) have resulted in the discovery of ERW1041E, a moderately potent inhibitor of TG2, which has found utility as a tool compound to study TG2 biology. As such, it has been shown that this inhibitor is capable of blocking the catalytic activity of TG2 in cell culture (Dafik and Khosla, 2011 Chem. Biol. 18, 58-66), in poly-I:C induced intestinal injury in mice (Dafik et al., 2012 PLoS One 7, e30642) and in the hypoxia-induced model of murine pulmonary hypertension (Diraimondo et al., 2013 ACS Chem. Biol.) In the latter study, it was also demonstrated that the inhibitor is well tolerated during twice daily administration for several weeks.
These studies showed the promise of DHI-based inhibitors in vivo, however there are several drawbacks of ERW1041E that needed to be addressed in a clinical lead compound, namely its moderate potency and its lack of selectivity. A higher potency would allow a reduction in the dose of the TG2 inhibitor and thus reduce the chance of off-target effects. Regarding its selectivity, a clinical lead compound would need to be selective for TG2 versus catalytically active transglutaminases in humans. Crossreactivity of a clinical lead with the epidermal transglutaminase 1 (TG1) and the fibrin-stabilizing Factor XIIIa (FXIIIa) would be particularly undesirable, given that loss-of-function mutations in either genes give rise to stark disease-phenotypes in humans (see e.g. Klöck et al., 2012 Semin. Immunopathol. 34, 513-522). The present invention addresses the need for improved, clinically useful compounds.